Cell-cell fusion is fundamental to the development and physiology of multicellular organisms, but little is known of its mechanistic underpinnings. Recent studies in several model systems have begun to reveal fundamental principles underlying cell-cell fusion. In particular, studies in the fruit fly Drosophila have revealed an essential function of the actin cytoskeleton in myoblast fusion, the process in which mononucleate myoblasts fuse to form multinucleate muscle fibers. Specifically, we have revealed a cell type-specific, F-actin-enriched podosome-like structure that invades the opposing fusion partner with multiple protrusive fingers, which ultimately leads to fusion pore formation. In addition, studies in the round worm C. elegans have identified a pair of putative fusogenic proteins that are both necessary and sufficient to induce fusion in the embyro. Moreover, it has been shown that expressing the worm fusogens in a heterologous insect cell line, Sf9 cells can induce a low frequency of cell-cell fusion. We have now established a high-efficiency, inducible cell culture system by co-expressing the worm fusogen and a fly cell adhesion molecule in a Drosophila cell line that does not normally undergo fusion. Such co-expression results in a >10 fold increase in cell fusion efficiency compared with cells expressing the fusogen alone. We show that similar podosome-like structures are used in cultured cells to promote cell-cell fusion and that the Arp2/3 nucleation promoting factors are required for fusing cultured cells as for muscle cells in Drosophila embryos. Thus we have established a cell culture system that closely mimics myoblast fusion in vivo. The goal of this project is to further characterize the molecular and cellular mechanisms of this cell culture system and to use it as a tool to discover new genes involved in cell-cell fusion.